There exist various diseases for warm-blooded organisms, human or animal, wherein the number of different-type blood cells is reduced or insufficiently active compared to the healthy state.
A first such group of diseases is a pathologically reduced T cell count, in particular CD4 T cell count commonly, such as for AIDS or leukemic diseases in the course of chemotherapy or radiation therapy.
A second group of diseases, where the number or the activity of a certain sub-group of T cells maintaining the immunological tolerance for a healthy person, is reduced or has a disturbed function, comprises autoimmune-inflammatory diseases. Examples are rheumatic arthritis, inflammatory intestinal diseases, diabetes requiring insulin treatment, multiple sclerosis and the Guillain-Barré syndrome.
A third group of diseases is called granulocytopenia (e.g. neutropenia) or monocytopenia and relates to the granulocytes. Reasons for this disease may be: i) reduced granulocytopoiesis or monocytopoiesis (aplastic disturbance) because of bone marrow damages, for instance by chemical substances such as benzene, drugs such as cytostatics, immune suppressants, AZT and/or chloramphenicol (dose-dependent, toxic) or phenylbutazone, gold compounds, rarely chloramphenicol (dose-independent by pharmakinetic reactions), radiation or autoantibodies against stem cells (in some cases of immune neutropenia), because of bone marrow infiltration (leukemias, carcinomas, malignant lymphomas) and/or because of osteomyelosclerosis, ii) maturation disturbances of the granulocytopoiesis, for instance by congenital maturation disturbances of the myelopoiesis, Kostmann's syndrome (maturation stop of the myelopoiesis in the stage of the promyelocytes), cyclic neutropenia, myelodysplasia syndrome, vitamin B12 or folic acid deficiency with ineffective granulo-, erythro- and/or thrombopoiesis. Usual therapies comprise the administration of growth factors of the granulocytopoiesis (for instance G-CSF and GM-CSF).
A fourth group is called thrombocytopenia. Reasons may be: i) reduced thrombocytopoiesis in the bone marrow (aplastic disturbance or reduced megakaryocyte count in the bone marrow) because of bone marrow damages, for instance by chemical substances such as benzene, drugs such as cytostatics and/or immune suppressants, radiation or infections such as HIV, or autoantibodies against megakaryocytes (in some cases of immune thrombocytopenia), because of bone marrow infiltration (leukemias, carcinomas, malignant lymphomas) and/or because of osteomyelosclerosis, ii) maturation disturbances of the megakaryocytes (megakaryocytes in the bone marrow normal or increased) with ineffective thrombo-, erythro- and/or granulopoiesis with megaloblasts, giant rods etc. because of vitamin B12 or folic acid deficiency. Usual therapies comprise the omission of suspicious-appearing drugs, thrombocyte substitution (in case of disturbance of generation in the bone marrow: thrombopoietin) and MGDF (stimulation of the proliferation and maturation of megakaryocytes).
The fifth group are the aplastic anemias or bone marrow failure with aplasia/hypoplasia of the bone marrow and pancytopenia (stem cell disease). A congenital aplastic anemia is for instance the Fanconi's anemia. More frequent are the acquired aplastic anemias, such as the idiopathic aplastic anemia (reason unknown) and secondary aplastic anemias by drugs, toxic substances, ionizing radiation and virus infections (see above). Supportive therapy approaches comprise the substitution of erythrocytes/thrombocytes. Causal therapy approaches comprise the bone marrow transplantation or stem cell transplantation, immunosuppressive therapies (e.g. ATG) and other therapy measures such as administration of cytokines (GM-CSF, G-CSF, MGDF, and/or thrombopoietin).
Finally, in the course of the acute leukemia, there is often anemia, thrombocytopenia and/or granulocytopenia. Therapies comprise the substitution of erythrocytes and thrombocytes as required or the excitation of granulopoiesis by G-CSF and/or GM-CSF, the chemotherapy and bone marrow and/or stem cell transplantation.
It is common to the above diseases of the first and second groups that the respective blood cells carry CD28 on their surfaces. It is common to the third to sixth groups that the concerned blood cells in contrast do not carry CD28 on their surfaces.
For a better understanding of the invention, further the following technological background is important. The activation of resting T cells for the proliferation and functional differentiation requires firstly the occupation of two surface structures, so-called receptors: 1. of the antigen receptor having a different specificity from cell to cell and being necessary for the recognition of antigens, e.g. viral fission products; and of the CD28 molecule equally expressed on all resting T cells with the exception of a sub-group of the CD28 T cells of man, this molecule naturally binding to ligands on the surface of other cells of the immune system. This is the costimulation of the antigen-specific immune reaction by CD28. In a cell culture, these processes can be imitated by occupation of the antigen receptor and of the CD28 molecule with suitable mAbs. In the classic system of the costimulation, neither the occupation of the antigen receptor nor that of the CD28 molecule alone will lead to the T cell proliferation, the occupation of both receptors is however effective. This observation was made for T cells of man, mouse and rat.
There are however also known CD28-specific mAbs, which alone can induce the T cell proliferation. Such a superagonistic activation, i.e. independent from the occupation of the antigen receptor, of resting T lymphocytes by CD28-specific mAbs was found in the following systems: in the document Brinkmann et al., J. Immunology, 1996, 156: 4100-4106, it has been shown that a very small portion (5%) of human T lymphocytes, which because of the missing surface marker CD45 RO could be assigned to the group of the resting T lymphocytes, is activated by the CD28-specific mAb 9.3 normally requiring costimulation when adding the growth factor interleukin-2 (IL-2) without occupation of the antigen receptor. In the document Siefken et al., Cellular Immunology, 1997, 176: 59-65, it has been shown that a CD28-specific mAb prepared in a conventional way, i.e. by immunization of mice with human T cells, can activate in a cell culture a subgroup of human T cells without occupation of the antigen receptor for the proliferation, if CD28 is occupied by this mAb and the cell-bound mAbs are additionally crosslinked with each other by further antibodies. It is common to the in so far known antibodies that only a small portion of the T cells can be activated.
In the document Tacke et al., Eur. J. Immunol., 1997, 27: 239-247, two kinds of CD28-specific monoclonal antibodies with different functional properties have been described; costimulatory mAbs, which costimulate the activation of resting T cells with simultaneous occupation of the antigen receptor only; and superagonistic mAbs, which can activate without occupation of the antigen receptor T lymphocytes of all classes in vitro and in the test animal for proliferation. Both in so far known mAbs originate from an immunization with cells, on which rat CD28 is expressed, and are obtainable by different selections directed toward their respectively described properties. Finally, from the document WO 98/54225, another human CD28-specific superagonistic mAb is known in the art, namely CMY-2.
Surprisingly however, according to the document DE-100 50 935 A1, blood cells not carrying CD28 can also be stimulated with superagonistic CD28-specific mAbs with in vivo applications.
The in so far known superagonistic mAbs meet all requirements with regard to their stimulatory effects, it would however be desirable to need less mAbs for a defined stimulatory effect. Furthermore, the stimulation of T lymphocytes by superagonistic CD28-specific mAbs is up to now limited to two systems each having drawbacks: isolated T lymphocytes can often be stimulated in a cell culture by such mAbs only, if the culture dishes have before been coated with antibodies reacting with the used superagonistic mAbs and secondarily crosslinking. When using unseparated preparations of peripheral blood cells also containing T lymphocytes, and with in vivo application, the superagonistic mAbs may also be used as a soluble substance; the reason for this is presumably that the secondary crosslinking takes place by so-called Fc receptors on the non-T lymphocytes reacting with the constant portion of the superagonistic mAbs, the so-called Fc portion. This dependence from Fc receptors is problematic, inter alia, because of the variable portion of Fc receptor-positive cells in preparations of peripheral blood cells and because of the variability of the antibody binding capacity caused by different Fc receptor alleles. Furthermore, the repeated addition of Fc receptor-expressing non-T lymphocytes is necessary for a repeated in vitro stimulation of T lymphocytes by soluble superagonistic mAbs. For use with human beings, this requires additional logistic and safety measures.
It is well known to multiply T cells in a culture by using small beads to which are bound CD28-specific costimulatory mAbs as well as TCR (CD3)-specific mAbs. Here there is a first drawback that two different substances are required. This requires a considerable effort when preparing under GMP conditions, which are legally required for preparations intended for therapy. Further, it is disadvantageous that each of these substances needs to be used in a relatively high concentration, since due to the common immobilization not all the molecules are available on a bead for the necessarily simultaneous binding of target cells, not for steric reasons alone.